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Abstract

Background

Chemotherapeutic treatment results in chronic pain in an estimated 30-40 percent of
patients. Limited and often ineffective treatments make the need for new therapeutics
an urgent one. We compared the effects of prophylactic cannabinoids as a preventative
strategy for suppressing development of paclitaxel-induced nociception. The mixed
CB1/CB2 agonist WIN55,212-2 was compared with the cannabilactone CB2-selective agonist AM1710, administered subcutaneously (s.c.), via osmotic mini pumps
before, during, and after paclitaxel treatment. Pharmacological specificity was assessed
using CB1 (AM251) and CB2 (AM630) antagonists. The impact of chronic drug infusion on transcriptional regulation
of mRNA markers of astrocytes (GFAP), microglia (CD11b) and cannabinoid receptors
(CB1, CB2) was assessed in lumbar spinal cords of paclitaxel and vehicle-treated rats.

Results

Both WIN55,212-2 and AM1710 blocked the development of paclitaxel-induced mechanical
and cold allodynia; anti-allodynic efficacy persisted for approximately two to three
weeks following cessation of drug delivery. WIN55,212-2 (0.1 and 0.5 mg/kg/day s.c.)
suppressed the development of both paclitaxel-induced mechanical and cold allodynia.
WIN55,212-2-mediated suppression of mechanical hypersensitivity was dominated by CB1 activation whereas suppression of cold allodynia was relatively insensitive to blockade
by either CB1 (AM251; 3 mg/kg/day s.c.) or CB2 (AM630; 3 mg/kg/day s.c.) antagonists. AM1710 (0.032 and 3.2 mg/kg /day) suppressed
development of mechanical allodynia whereas only the highest dose (3.2 mg/kg/day s.c.)
suppressed cold allodynia. Anti-allodynic effects of AM1710 (3.2 mg/kg/day s.c.) were
mediated by CB2. Anti-allodynic efficacy of AM1710 outlasted that produced by chronic WIN55,212-2
infusion. mRNA expression levels of the astrocytic marker GFAP was marginally increased
by paclitaxel treatment whereas expression of the microglial marker CD11b was unchanged.
Both WIN55,212-2 (0.5 mg/kg/day s.c.) and AM1710 (3.2 mg/kg/day s.c.) increased CB1 and CB2 mRNA expression in lumbar spinal cord of paclitaxel-treated rats in a manner blocked
by AM630.

Conclusions and implications

Cannabinoids block development of paclitaxel-induced neuropathy and protect against
neuropathic allodynia following cessation of drug delivery. Chronic treatment with
both mixed CB1/CB2 and CB2 selective cannabinoids increased mRNA expression of cannabinoid receptors (CB1, CB2) in a CB2-dependent fashion. Our results support the therapeutic potential of cannabinoids
for suppressing chemotherapy-induced neuropathy in humans.

Keywords:

Background

Cannabinoids attenuate or, in some cases, prevent pain associated with surgery [1], inflammation [2], internal organs [3], and neuropathies (for review see [4]). Neuropathic pain is associated with abnormal changes in the peripheral and/or central
nervous system resulting in non-adaptive, chronic pain. Clinical manifestations of
neuropathic pain are notoriously unresponsive to traditional analgesics. Chemotherapeutic
treatment with antineoplastic agents, while effective at eliminating harmful malignancies,
is also associated with severe side effects. Of these side effects, emesis, alopecia,
and myelosuppression have received the spotlight; however, a new front runner has
recently emerged. Neuropathic pain associated with chemotherapeutic treatment is dose-limiting
and a major factor influencing discontinuation of treatment [5,6]. Chemotherapy-induced neuropathy is positively correlated with cumulative chemotherapeutic
dose [7], and affected patients are more likely to experience other neuropathies [8]. An aging US population, coupled with diagnostic and medical advances in cancer treatment,
means that more cancer survivors will be impacted by, and living longer with, chemotherapy-induced
neuropathy. Thus, identification of prophylactic treatments that block development
of chemotherapy-induced neuropathy represents an urgent medical need.

Chemotherapeutic agents are divided into three mechanistically distinct classes. These
classes include the vinca alkaloids, platinum-derived agents, and taxanes. Taxanes
(e.g., paclitaxel, docetaxel) produce antineoplastic effects by stabilizing microtubules
through binding to β-tubulin, thereby disrupting normal cell mitosis and triggering
the mitochondrial apoptosis pathway [9]. Paclitaxel is a preferred agent for treatment of ovarian, breast, and lung cancers;
however, a high percentage of patients experience neuropathic pain – a type of pain
poorly treated with available drugs [10]. Mechanisms underlying development of paclitaxel-induced neuropathy remain incompletely
understood but may involve changes in glial activation [11,12].

Cannabinoid agonists suppress paclitaxel-induced neuropathic nociception in animal
models through activation of both CB1[13] and CB2[14-16] cannabinoid receptor subtypes. Our laboratory first demonstrated CB2 receptor-mediated suppression of neuropathic allodynia induced by chemotherapeutic
treatment with vincristine [17], paclitaxel [14,18], and cisplatin [15]. Previous prophylactic treatment strategies with cannabinoids in a traumatic nerve
injury model demonstrated that pre-emptive cannabinoids produced greater antinociception
relative to post-injury treatment [19]. Here we investigate the therapeutic efficacy of prophylactically administered WIN55,212-2,
a mixed cannabinoid (CB1/CB2) agonist, and AM1710, a CB2-preferring agonist, on the development of chemotherapy-induced neuropathy in the
paclitaxel model. Osmotic mini pumps were used to continuously infuse cannabinoids
before, during, and after paclitaxel treatment, to emulate a prophylactic analgesic
strategy achievable in clinical oncology settings. We compared development of mechanical
and cold allodynia, both common clinical manifestations of paclitaxel-induced neuropathy
[10,20]. We hypothesized that chronic prophylactic cannabinoid infusion would produce sustained
suppression of paclitaxel-induced behavioral sensitization to mechanical and cold
stimulation. Furthermore, we evaluated whether long-term transcriptional changes in
mRNA markers of astrocytes (GFAP), microglia (CD11b), and cannabinoid receptors (CB1, CB2) would accompany long lasting anti-allodynic efficacy of cannabinoids.

Results

General results

Paclitaxel-treated animals showed reduced sensitivity to heat on day 6 (F1,10 = 20.745, P < 0.01; Figure 1a), but not at subsequent time points (P > 0.16), while the same animals developed hypersensitivity to mechanical stimulation
(i.e., mechanical allodynia) (F1,10 = 6.191, P < 0.05; Figure 1b). Based upon these results, animals implanted with osmotic pumps were evaluated
for responsiveness to mechanical and cold stimulation only.

Osmotic mini pump dispersion volume was calculated by subtracting the fill volume
from the residual volume in the pump reservoir following pump removal (day 22). The
pump dispersion volume differed between groups in which drugs were dissolved in the
DMSO:PEG400 vehicle (F19,180 = 2.213, P < 0.01). Post-hoc analysis revealed that pump dispersion volume for the Taxol-WIN55,212-2
(1 mg/kg/day s.c.) group was less than half (< 43%) of other groups dissolved in the
same vehicle. No other differences were found. Mechanical withdrawal thresholds did
not differ between either the right or left paw on any given day for animals tested
up to 20 (P > 0.98) or 50 (P > 0.71) days post-chemotherapy treatment; therefore, withdrawal thresholds are presented
as the mean of duplicate measurements, averaged across paws. Two dependent measures
for cold allodynia were evaluated: percentage of paw withdrawals and duration of paw
withdrawal. Duration of paw withdrawal in response to topical acetone application
is a reported measure of cold allodynia [21-23]. However, we found this measure highly variable in rat subjects (data not shown)
and consequently only the percentage of paw withdrawals is reported here. Percentage
of paw withdrawals to cold stimulation did not differ between either paw on any given
day for animals tested up to 21 (P > 0.33) or 51 (P > 0.82) days post-paclitaxel; therefore, the percentage of paw withdrawals is presented
as the mean of duplicate measurements averaged across paws.

To control for any possible effects associated with the vehicle used to dissolve cannabinoids
(DMSO:PEG 400 in a 1:1 ratio), a subset of animals treated with either paclitaxel
or cremophor received saline in their osmotic mini pumps. No differences were detected
between paclitaxel-treated animals that received vehicle (DMSO:PEG 400; n = 14) or
saline (n = 4) in any behavioral parameter assessed (i.e., mechanical threshold, cold
withdrawal frequency, and locomotor activity). Similarly, no differences were noted
between cremophor-treated animals receiving chronic infusions of vehicle (DMSO:PEG
400; n = 8) or saline (n = 4). Therefore, vehicle and saline groups were combined
for each condition and are referred to as the Taxol-vehicle group and cremophor-vehicle
group, respectively.

Anti-allodynic effects of the mixed CB1/CB2 agonist WIN55,212-2

Paclitaxel-treated animals receiving vehicle infusions developed mechanical allodynia
relative to cremophor-treated counterparts; mechanical allodynia was apparent on day
2 and persisted until the final test day prior to pump removal (day 20) (F48,660 = 3.880, P < 0.001; P < 0.01 for each comparison; Figure 2c). WIN55,212-2 (0.1 mg/kg/day s.c.) produced a transient antinociceptive effect prior to paclitaxel treatment on day -2 (P < 0.05); this antinociceptive effect was observed relative to paclitaxel-treated
groups receiving either vehicle or WIN55,212-2 (1.0 mg/kg/day s.c.). WIN55,212-2 (0.5 mg/kg/day s.c.)
blocked development of paclitaxel-induced mechanical allodynia (F4,55 = 32.964, P < 0.001; Figure 2c) and normalized mechanical thresholds relative to the Taxol-vehicle group at all
time points (P < 0.05 for each comparison). WIN55,212-2 (0.1 mg/kg/day s.c.) also suppressed the
development of paclitaxel-evoked mechanical allodynia over the time course corresponding
to drug delivery (P < 0.05 for each comparison) but failed to normalize thresholds relative to cremophor-vehicle
levels.

Anti-allodynic effects of the CB2 agonist AM1710

AM1710 (3.2 and 0.032 mg/kg/day s.c.) blocked development of paclitaxel-evoked mechanical
allodynia (F4,59 = 41.988, P < 0.001; Figure 2d) over the time course corresponding to drug delivery (F48,708 = 5.186, P < 0.001; P < 0.01 for each comparison). AM1710 (3.2 and 0.032 mg/kg/day s.c.) increased mechanical
withdrawal thresholds relative to the Taxol-vehicle group beginning on day 4 and this
effect was maintained for the duration of the study (P < 0.05 for each comparison). The high dose of AM1710 (3.2 mg/kg/day s.c.) preferentially
increased mechanical paw withdrawal thresholds relative to the middle dose (0.32 mg/kg/day s.c.)
from days 12–20 (P < 0.05 for each comparison). Moreover, AM1710 (3.2 mg/kg/day s.c) normalized paw
withdrawal thresholds in paclitaxel-treated animals to those observed in the cremophor-vehicle
group at all time points.

Anti-allodynic effects of the mixed CB1/CB2 agonist WIN55,212-2

Paclitaxel-induced cold allodynia developed by day 5 and was stable until the final
test day associated with drug delivery (day 21) (F20,275 = 7.197, P < 0.001; P < 0.05 for each comparison; Figure 2e). The middle and low doses of WIN55,212-2 (0.5 and 0.1 mg/kg/day s.c.) blocked development
of cold allodynia in paclitaxel-treated animals (F4,55 = 11.428, P < 0.001, P < 0.05 for each comparison; Figure 2e) for the duration of drug delivery. The high dose of WIN55,212-2 (1 mg/kg/day s.c.)
failed to fully suppress development of paclitaxel-induced cold allodynia. However,
animals in this group nonetheless showed protection against cold allodynia relative
to paclitaxel-vehicle treated animals at some observation intervals (i.e., days 11
and 21; P < 0.001).

Discussion

Prophylactic administration of cannabinoid analgesics protected against the development
of paclitaxel-induced hypersensitivities to mechanical and cold stimulation in a preventative
fashion. Both the mixed cannabinoid CB1/CB2 agonist WIN55,212-2 and the CB2 agonist AM1710 blocked development of paclitaxel-induced mechanical and cold allodynia.
Strikingly, the protective prophylactic effects of both WIN55,212-2 and AM1710 were
preserved following drug removal, with the CB2-specific agonist providing a longer duration of protection against allodynia development
for both mechanical and cold modalities. In our study, paclitaxel produced marked
mechanical and cold allodynia but not heat hyperalgesia, as observed in a different
dosing paradigm (cumulative dose: 4 mg/kg i.p.) [28]. In vehicle (cremophor) treated controls, the most efficacious doses of these cannabinoids
also failed to produce antinociception, suggesting that cannabinoids were anti-allodynic
rather than analgesic under these conditions.

WIN55,212-2 (0.5 and 0.1 mg/kg/day s.c.) suppressed the development of paclitaxel-induced
mechanical and cold allodynia both during drug delivery and following drug removal.
Our study is the first to evaluate duration of efficacy, dose response, and pharmacological
specificity of prophylactic WIN55,212-2. Anti-allodynic effects of both doses were
present 11 (mechanical) and 12 (cold) days following cessation of drug delivery. WIN55,212-2
(0.5 mg/kg/day s.c.)-induced suppression of mechanical hypersensitivity was dominated
by CB1 receptor activation because anti-allodynic efficacy was blocked by AM251. The CB2 antagonist AM630 (3 mg/kg/day s.c.) prevented anti-allodynic efficacy of AM1710 but
failed to eliminate WIN55,212-2-mediated anti-allodynia. Interestingly, blockade of
WIN55,212-2-mediated anti-allodynic effects to cold was not achieved with either antagonist.
However, the same antagonist infusion conditions blocked either WIN55,212-2 mediated
suppression of mechanical allodynia (AM251) or AM1710-mediated suppression of both
mechanical and cold allodynia (AM630), documenting efficacy of antagonist infusion
conditions employed here.

We could find only one report of WIN55,212-2-induced suppression of cold allodynia
in a neuropathic pain model (spinal nerve ligation) where pharmacological specificity
was assessed; anti-allodynic effects were blocked by a CB1 (SR141716a) but not a CB2 (SR144528) antagonist [29]. Blockade of both CB1 and CB2 receptors may be required to fully prevent anti-allodynic effects of WIN55,212-2.
However, limitations in compound solubility prohibited co-administration of both antagonists
in one pump.

Few studies have examined cannabinoid-mediated modulation of cold allodynia and/or
its development in neuropathic pain models and more work is necessary to determine
functional contributions of each receptor. WIN55,212-2 (0.5 mg/kg/day s.c.) treatment
increased both CB1 and CB2 receptor mRNA expression within lumbar spinal cord of paclitaxel-treated animals
on day 22, an effect blocked by concurrent AM630 (3 mg/kg/day s.c.) treatment. WIN55,212-2
also ameliorates established paclitaxel-induced nociception [13] and repeated administration (1 mg/kg i.p. × 14 days) prevents nociception development
during drug delivery [11].

Strikingly, doses of AM1710 as low as 0.032 mg/kg/day blocked the development of paclitaxel-induced
allodynia in our study and these effects were preserved for approximately three weeks
following cessation of drug delivery. Prophylactic AM1710 treatment suppressed development
of both paclitaxel-induced mechanical and cold allodynia, with high (3.2 mg/kg/day s.c.)
and low (0.032 mg/kg/day s.c.) doses exhibiting the greatest efficacy. A similar U-shaped
dose response curve was obtained for thermal antinociception (plantar test) in naive
animals without observable CB1-mediated side effects [30].

Activity meter assessments conducted during prophylactic treatment (day 19) and following
drug removal (day 31), failed to reveal major differences between groups. Thus, chronic
infusion of either the mixed CB1/CB2 agonist or the CB2 agonist was unlikely to nonselectively activate CB1 receptors; no evidence for hypoactivity [35], a cardinal sign of CB1 activation, was observed. These findings are consistent with the results documenting
absence of cardinal signs of CB1 receptor activation following acute administration of AM1710 [30].

Glial activation mediates alterations in synaptic transmission for a number of excitatory
and inhibitory mediators known to be important for the maintenance of neuropathic
pain states [36]. Because of the prolonged suppression of paclitaxel-induced neuropathy after removal
of cannabinoid agonists, we chose to analyze transcriptional changes in markers of
glial activation. GFAP mRNA expression in lumbar spinal cord on day 22 (i.e., approximately
24 h after the pump ceased to release drug) showed a trend toward increased expression
in paclitaxel- relative to cremophor-treated controls, while no change in CD11b expression
was observed. Increases in astrocytic activation (GFAP) with no corresponding changes
in microglial activation (OX42, Iba1, and phosphorylated p38) were also recently observed
with the same paclitaxel-induced neuropathy dosing protocol used here [12]. In another study, paclitaxel failed to produce microglial activation (% of cremophor-control
staining) on day 27 post-treatment [37]. By contrast, MDA7 and WIN55,212-2 suppressed paclitaxel-induced glial activation
(on days 28 and 29 post-treatment, respectively) when immunohistochemical staining
for astrocytes (GFAP) and microglia (CD11b) was compared with naive animals [11], and it remains unclear whether vehicle or cremophor administration alters glial
activation [16]. Cremophor can produce side effects in both clinical use and animal models [38], and assumptions that it is inert are not appropriate.

Prophylactic treatment with either a mixed CB1/CB2 agonist or a CB2 agonist, while failing to produce robust alterations in lumbar spinal cord glial
expression, increased CB1 and CB2 mRNA expression. This effect was blocked by CB2 receptor blockade. Upregulation of endocannabinoids and cannabinoid receptors is
associated with several neuropathic pain models (for review see [39]). However, to our knowledge, very few, if any, studies have evaluated alterations
following prophylactic cannabinoid treatment. Increased receptor densities could increase
the potency or efficacy of prophylactic cannabinoids in this model. More work is necessary
to determine whether changes in CB1 and CB2 mRNA levels observed here are also associated with changes in receptor protein. Alternatively,
increased CB1 and CB2 mRNA expression could reflect compensatory changes in transcription following chronic
agonist-induced downregulation of receptors. More work is necessary to fully characterize
the duration of these effects and their therapeutic implications.

Translation to the clinic

Our preclinical studies [14,15,17,18] motivated completion of a pilot clinical trial utilizing Sativex, an oromucosal extract
containing Δ9-tetrahydrocannabinol and cannabidiol, for treatment of chemotherapy-induced neuropathy.
Sativex suppressed established chemotherapy-induced neuropathic pain in a subset of
responders (5 of 18) in this double-blind placebo-controlled crossover pilot [40], supporting a further evaluation of the clinical viability of cannabinoid-based pharmacotherapy.

Conclusion

Prophylactic treatment has been tested as a preventive strategy for paclitaxel-induced
neuropathic nociception with multiple drugs (for review see [41]). Here, we demonstrate that cannabinoid agonists with different mechanisms of action
prevent development of paclitaxel-induced neuropathic nociception during treatment
and approximately two to three weeks following cessation of drug delivery. Paclitaxel
treatment marginally altered long-term GFAP mRNA expression in lumbar spinal cord
and this expression was unaffected by prophylactic cannabinoids, whereas CD11b mRNA
expression was unchanged. Prophylactic treatment with either WIN55,212-2 (0.5 mg/kg/day s.c.)
or AM1710 (3.2 mg/kg/day s.c.) in paclitaxel animals did, however, increase both CB1 and CB2 receptor mRNA expression, an effect blocked by concurrent AM630 (3 mg/kg/day s.c.)
administration. Some inroads have been made toward discovering mechanisms for cannabinoid-mediated
suppression of paclitaxel-induced neuropathy, but more work is necessary to determine
the scope and time course of this complex interaction. Our study suggests that further
clinical cannabinoid trials [40] for chemotherapy-induced peripheral neuropathy are warranted.

Methods

Subjects

Rats

One hundred seventy-six adult male Sprague–Dawley rats (beginning weight: 300-400 g;
Harlan, Indianapolis, IN) were used in these experiments. All procedures were approved
by the University of Georgia Animal Care and Use Committee and followed the guidelines
for the treatment of animals of the International Association for the Study of Pain.
Animal experiments were conducted in full compliance with local, national, ethical
and regulatory principles, and local licensing regulations of the Association for
Assessment and Accreditation of Laboratory Animal Care (AAALAC) International’s expectations
for animal care and use/ethics committees.

Animals were allowed a minimum of one week habituation prior to beginning the study.
Animals were single housed and maintained in a temperature (70-72 °F ± 4 °F) and humidity
(30-70%) controlled facility on a 12 hour light cycle (lights on: 07:00 and lights
off: 19:00). Food and water was available ad libitum. Following the initial pilot
study (n = 17), all animals with osmotic mini pumps were allowed nyla bones (BioServe;
Frenchtown, NJ) due to the study duration. Corn cob bedding containing metabolized
paclitaxel was treated as chemical hazard waste and disposed of according to appropriate
institutional guidelines.

Drugs and chemicals

Paclitaxel (Taxol) was obtained from Tecoland (Edison, NJ). Polyethylene Glycol 400
(PEG 400) was purchased from VWR International (West Chester, PA). Acetone was purchased
from J.T. Baker (Phillipsburg, NJ). Cremophor EL, Dimethyl Sulfoxide (DMSO), and WIN55,212-2
((R)-(+)-[2,3-Dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone
mesylate salt) were obtained from Sigma Aldrich (St. Louis, MO). AM1710 (3-(1’,1’-dimethylheptyl)-1-hydroxy-9-methoxy-6H-benzo[c]chromene-6-one), AM251 (N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide), and AM630 (6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone (Iodopravadoline) were synthesized in the Center
for Drug Discovery by one of the authors (by GT, VKV, and AZ respectively). Rat subjects
received paclitaxel dissolved as previously described [42], administered in a volume of 1 ml/kg. Briefly, paclitaxel was dissolved in a 1:2
ratio of working stock (1:1 ratio of cremophor EL and 95% ethanol) to saline. AM1710,
WIN55,212-2, AM251, and AM630 were dissolved in a vehicle of DMSO:PEG 400 in a 1:1
ratio. The selected vehicle was the most compatible for dissolving cannabinoids to
be used in Alzet osmotic mini pumps with no reported adverse side effects [43-45].

General experimental methods

In an initial study, animals were evaluated for development of paclitaxel-induced
behavioral sensitization to mechanical and heat stimulation. Responsiveness to different
modalities of cutaneous stimulation was assessed on alternate days to avoid sensitization.
All subsequent studies used animals surgically implanted with osmotic mini pumps.
Baseline assessments of withdrawal thresholds to mechanical and cold (acetone drops)
stimulation of the hind paw occurred 48 h (day -8) and 24 h (day -7) prior to surgery,
respectively. Osmotic mini pumps (Alzet model 2ML4, Cupertino, CA) were implanted
subcutaneously through an incision between the scapulae. Responsiveness to mechanical
and cold stimulation was reassessed post-surgery (i.e., after pump implantation but
within 48 h prior to initiation (on day 0) of paclitaxel dosing). Animals were weighed
on all testing and surgical/sacrifice dates. A subset of animals was sacrificed via
live decapitation (day 22) to extract lumbar spinal cords. Certain groups (e.g., antagonist
alone conditions, submaximal doses of agonists, cremophor-agonist groups) were only
tested through day 22. Osmotic mini pumps were removed in all remaining animals (day
22), and following a short recovery period, responses to mechanical and cold stimulation
were reassessed until day 51 post-paclitaxel.

Drug doses were estimated based on the peak osmotic mini pump performance reported
by the manufacturer (2.5 μl/hr) and an average rat weight of 375 grams. A small percentage
of animals (4.2%) presented with edema around the pump site (seromas). Alzet reports
this side effect in < 5% of animals. Treatment for these animals was supervised by
the attending veterinarian and consisted of draining fluid every 3 days, or as needed.
Six animals (3.6%) were re-sutured following surgery. One of the six animals developed
an infection and was treated (from days 16–22) with daily injections of an antibiotic
(Enrofloxacin 4.5 mg/ml, 0.4 cc s.c., 2× daily) and sterile water (1 cc s.c., 1× daily)
as prescribed by the staff veterinarian. One animal died during the first paclitaxel
injection and this animal was excluded from all analyses.

Behavioral measurements, surgeries, chemotherapeutic treatment, and tissue removal
were performed by a single experimenter (EJR). Coded testing sheets were used to preserve
blinding. Behavioral testing was performed in the presence of a white noise generator
to mask extraneous noise.

Surgical implantation and removal of osmotic mini pumps

Osmotic mini pumps were implanted under isoflurane anesthesia (Isoflo®, Abbott Laboratories,
Chicago, IL). The osmotic mini pump was inserted through a surgical incision made
between the scapulae; incisions were sutured closed. In the instances where two pumps
were implanted (i.e., agonist and antagonist co-administration conditions), pumps
were placed in the same pocket. The Alzet model 2ML4 pump has an approximate 2 ml
reservoir that releases a preloaded drug or vehicle at a rate of 2.5 ul/hr for approximately
28 days. The pump begins to release the preloaded drug approximately 4–6 hours after
implantation; the flow rate is not subject to variations in body temperature. Osmotic
mini pumps were weighed before and after being filled with drug or vehicle. The difference
of these two values provided an approximate pump fill volume. The animals were given
three days (days -5 through -3) to recover from surgery before testing resumed. Animals
were either sacrificed or underwent surgery on day 22 to remove pumps; this time point
corresponds to the 29th day following pump implantation, at which point the pump should
have released its contents. Following pump removal, the residual pump volume was estimated
by withdrawing the remaining fluid within the pump reservoir. Animals that underwent
surgical removal of osmotic mini pumps were allowed three days of recovery (days 23–25)
prior to resumption of behavioral testing.

Assessment of paw withdrawal latencies to heat stimulation

Paw withdrawal latencies to radiant heat were measured in duplicate for each paw using
the Hargreaves test [46] and a commercially available plantar stimulation unit (IITC model 336; Woodland Hills,
CA). Rats were placed underneath inverted plastic cages positioned on an elevated
glass platform and allowed a minimum of 20 min to habituate prior to testing. Radiant
heat was presented to the hind paw midplantar region through the floor of the glass
platform. The intensity of the heat source was adjusted such that an average baseline
latency of approximately 20 sec was achieved [47]. Stimulation was terminated upon paw withdrawal or after 40 s to prevent tissue damage.
Approximately 4 minute interstimulation intervals were allowed between tests. Thermal
withdrawal latencies were evaluated before (day 0) and on days 2, 6, 10, 14 and 18
following initiation of paclitaxel dosing. The same animals were tested for the development
of mechanical allodynia. Baseline responses to mechanical stimulation (methodology
below) were measured (on day 0) before baseline responses to thermal stimulation.
A minimum of 1 hour was allowed to elapse between baseline measurements.

Assessment of mechanical withdrawal thresholds

Mechanical withdrawal thresholds were assessed using a digital Electrovonfrey Anesthesiometer
(IITC model Alemo 2390–5; Woodland Hills, CA) equipped with a rigid tip. Rats were
placed underneath inverted plastic cages positioned on an elevated mesh platform and
allowed a 20 min habituation period prior to testing. Stimulation was applied to the
hind paw midplantar region through the floor of a mesh platform. Mechanical stimulation
was terminated upon paw withdrawal; consequently, no upper threshold limit was set
for termination of a trial. Two thresholds were taken for each paw. Approximately
2 minute interstimulation intervals were allowed between tests. Mechanical withdrawal
thresholds were measured on days 0, 4, 8, 12, 16 and 20 for animals that did not receive
osmotic mini pumps (Figure 1). Mechanical withdrawal thresholds were measured every 2–6 days (i.e., days -8, -2,
0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20) for all animals that received osmotic mini
pumps. A subset of osmotic mini pump animals were tested until day 50 (testing continued
with the following schedule: days 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,
and 50).

Assessment of cold allodynia

Cold allodynia was assessed using acetone drops applied to the hind paw midplantar
surface as previously described [15,48]. Rats were placed underneath inverted plastic cages positioned on an elevated mesh
platform and allowed a 20 min habituation period prior to testing. Acetone was loaded
into a one cc syringe barrel with no needle tip. One drop of acetone (approximately
20 μl) was applied through the mesh platform onto the hind paw midplantar surface.
Care was taken to gently apply the bubble of acetone to the skin without inducing
mechanical stimulation by syringe barrel contact with the paw.

Paw withdrawal was recorded as a binary response (presence or absence) and was frequently
accompanied by nocifensive behaviors (e.g., rapid flicking of the paw, chattering,
biting, and/or licking of the paw). These nocifensive behaviors were recorded as duration
of acetone response. Five measurements were taken for each paw. Testing order alternated
between paws (i.e., right, left). No cut-off latency was enforced. Approximately 2 min
interstimulation intervals were allowed between testing of right and left paws. A
minimum interstimulation interval of 5 min was allowed between testing each pair of
paws (right and left). Cold allodynia testing took place on days -7, -1, 5, 11, 17
and 21 for all animals with osmotic mini pumps. Five days were allowed between assessments
of cold allodynia to avoid hypersensitivity with one exception. Animals were tested
on day 21 because osmotic mini pumps would purportedly still be releasing drug (i.e.,
28 days following pump implantation). A subset of animals was tested to day 51 (i.e.,
testing for these animals continued with the following schedule: days 27, 33, 39,
45, and 51).

Locomotor activity

Total distance traveled (cm) was assessed using an activity monitor chamber (Coulbourn
Instruments, Whitehall, PA) measuring 40.64 cm3. The apparatus was housed in a darkened room and red light was used to provide illumination.
Tracking beams were positioned 2.54 cm apart giving 1.27 cm in spatial resolution.
Activity was automatically measured by computerized analysis of photobeam interrupts
(TruScan 2.0; Coulbourn Instruments, Whitehall, PA). Animals were allowed a minimum
of 15 minutes to habituate to the room prior to being placed undisturbed in the activity
meter for 15 min. Chlorhexidine was used to clean the activity meter after each animal.
Activity meter assessment took place both during (day 19) and following termination
(day 31) of drug delivery in a subset of animals that received chronic infusions.

Statistical analyses

Percentage of paw withdrawals from acetone application to the hind paws was calculated
using the following formula: ((Total number of paw withdrawals) * 100)/10. Data were
analyzed using analysis of variance (ANOVA) for repeated measures, one-way ANOVA,
or planned comparison t-test as appropriate. SPSS 19.0 (SPSS Incorporated, Chicago, IL, USA) statistical
software was employed. The Greenhouse-Geisser correction was applied to all repeated
factors where the epsilon value from Mauchly’s Test of Sphericity was < 0.75 and significance
level was P < 0.05. Degrees of freedom reported for interaction terms of repeated factors are
uncorrected values in cases where the Greenhouse-Geisser correction factor was applied.
Post-hoc comparisons between the primary control group (paclitaxel-vehicle) and other
experimental groups were performed using the Dunnett test (2-sided). Post-hoc comparisons
between different experimental groups were also performed to assess dose–response
relationships and pharmacological specificity using the Tukey test. Levene’s test
for homoscedasticity was applied to all planned comparison t-tests. P < 0.05 was considered statistically significant.

Abbreviations

Competing interests

Dr. Alexandros Makriyannis serves as a consultant for MAK Scientific. No other authors
declare competing interests.

Authors’ contributions

EJR contributed to experimental design, completed all surgeries, behavioral studies,
and tissue extractions, analyzed data and drafted the manuscript. LD isolated RNA,
carried out the RT-PCR studies and analyzed data. GAT synthesized AM1710. VKV synthesized
AM251 and AM630. AMZ synthesized AM630. YYL assisted with RT-PCR and contributed to
manuscript preparation and data interpretation. AM provided cannabinoid compounds
and contributed to data interpretation. AGH designed the study, participated in its
coordination and implementation, and wrote the manuscript with EJR. All authors read
and approved the final manuscript.

Acknowledgments

This work was supported by DA021644, DA028200, DA022478, and DA037673 (to AGH) and
DA9158, DA3801 (to AM). EJR was supported by an ARCS Foundation Fellowship, an APF
Graduate Fellowship, a Psi Chi Graduate Research Grant and a Sigma Xi Grant-in-Aid
of Research Award.